Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). The natural host of BLV is cattle and much like the case of HTLV-1 in humans, about ~5% of ... [more ▼]

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). The natural host of BLV is cattle and much like the case of HTLV-1 in humans, about ~5% of infected individuals develop leukemia/lymphoma following a long period of asymptomatic infection (~7 years in cattle, several decades in human). Experimental infection of sheep with BLV results in a reduced latency period (2 years on average), making for an attractive cancer model. A further advantage of the BLV system is that it is possible to infect sheep via injection of a cloned provirus, facilitating the mutation of specific parts of the viral genome to examine the function of viral products in vivo. Like HTLV-1, the BLV mRNAs/proteins are transcribed from the viral 5’ long terminal repeat (LTR), a region rich in regulatory elements. It was previously believed that the BLV provirus was transcriptionally silent in tumors, however we identified a cluster of five abundantly expressed non-canonical RNA polymerase III dependent microRNAs (miRNAs) encoded by BLV (Rosewick et al., PNAS 2013). In addition, using RNA sequencing we recently discovered viral antisense transcripts originating in the 3' Long Terminal Repeat (LTR) of the BLV provirus (Durkin et al., Retrovirology 2016) . While 5'LTR dependent transcription is absent in malignant cells, both the viral miRNAs and the antisense transcripts are expressed in all BLV induced leukemic and pre-leukemic samples examined to date, pointing to a vital role in the life cycle of the virus and a critical function in cellular transformation. [less ▲]

Background Adult T-cell leukemia/lymphoma (ATL) is an aggressive CD4+ T-cell malignancy caused by HTLV-1 infection and associated with extremely poor prognosis. In France, treatment strategies mainly include chemotherapy, antiviral therapy and allogeneic stem cell transplantation, based on clinical subtype and therapeutic responses. Response to treatment is evaluated based on consensus criteria defined in 2009 (Tsukasaki K, Hermine O et al, JCO 2009). Although immuno-phenotyping, TCRγ rearrangement and HTLV-1 proviral load (PVL) quantification are often assayed, complete clinical remission (CR) is currently defined by morphological and cytological criteria i.e. complete blood cell counts (CBC) and the presence of abnormal lymphocytes. Given the extremely poor prognosis and high rates of early relapse, a revision of the response criteria is required, calling for improved tools that integrate specific aspects of the pathophysiology of ATL to better estimate response to treatment. Methods We retrospectively analyzed longitudinal PBMC samples from 6 ATL patients diagnosed with a leukemic subtype (5 acute and 1 chronic-aggressive) which all achieved CR upon therapy, yet relapsed after a median time of 15,9 months (range 2,4-70,7). CR was assessed by morphological and cytological criteria. An improved high throughput sequencing (HTS) based method was utilized to map and quantify the abundance of HTLV-1 genomic integration sites (IS), overcoming some of the limitations of previously published protocols. The dynamic range was increased by assaying both the 5’LTR and 3’LTRs, allowing better determination of clone abundance and revealing 5’ deletions. An enrichment step limited PCR duplicates. The addition of off-the-shelf Illumina primers simplified library multiplexing and reduced the costs to the point where the protocol could be applied to a clinical setting. Results HTS- mapping of HTLV-1 IS at diagnosis revealed in all cases a unique IS that constituted 92-99% of proviral genomes, with PVLs of 33-510%. All patients were treated and achieved CR which was characterized by normalized CBC, <5% abnormal lymphocytes and the absence of measurable tumors for >4 weeks. For 3/6 patients, the clone frequency distribution of HTLV-1 infected cells at CR was composed of multiple low abundance clones, of which the unique presumed malignant IS contributed to less than 2% of proviral genomes. In contrast, clonality analysis of the remaining 3/6 patients revealed that the relative abundance of the malignant clone detected at diagnosis remained dominant at CR (36-83% of PVL), despite clinical response criteria typical of CR and a 3-20-fold decrease in PVLs. These patients relapsed after 2.4, 2.9 and 3.4 months respectively with a dominant malignant clone >95% while patients with a polyclonal architecture showed significantly longer CR (28, 59 and 71 months). Conclusions Our observations highlight the great heterogeneity within an identical CR group, underlining the need for revisiting response criteria for ATL. Our results call for the use of this improved HTS-based method to measure HTLV-1 clonality as a tool to better estimate response to treatment, predict relapse and guide therapeutic choices in the course of treatment. [less ▲]

We herein describe a new method to fine-map GWAS-identified risk loci based on the Bayesian Least Absolute Shrinkage Selection Operator (LASSO) combined with a Monte Carlo Markov Chain (MCMC) approach, and corresponding software package (BayesFM). We characterize the performances of BayesFM using simulated data, showing that it outperforms standard forward selection both in terms of sensitivity and specificity. We apply the method to the NOD2 locus, a well-established risk locus for Crohn's disease, in which we identify 13 putative independent signals. [less ▲]

We herein study genetic recombination in three dairy cattle populations from France, New-Zealand and the Netherlands. We identify 2,395,177 crossover (CO) events in sperm cells transmitted by 2,940 sires to 94,516 offspring, and 579,996 CO events in oocytes transmitted by 11,461 cows to 25,332 offspring. When measured in identical family structures, the average number of CO in males (23.3) was found to be larger than in females (21.4). The heritability of global recombination rate (GRR) was estimated at 0.13 in males and 0.08 in females. The genetic correlation was equal to 0.66, indicating that shared variants are influencing GRR in both genders. Haplotype-based genome-wide association studies revealed seven genome-wide significant QTL. Variants identified by next-generating sequencing in 5 Mb windows encompassing the QTL peaks were imputed in order to perform a sequence-based association analysis. For four QTLs, we identified missense mutations in genes known to be involved in meiotic recombination among the most significantly associated variants. Most of the identified mutations had significant effects in both genders with three of them accounting each for approximately 10% of the genetic variance in males (the allelic substitution effect being approximately equal to one additional CO per genome). Thus, a large fraction of the genetic variance is associated with missense mutations in genes known to be involved in meiotic recombination. Our results are very different from reports of recombination in other species. For instance, in human, recombination rate is higher in females, distinct variants affect recombination rate in males and females, and the genetic correlation is close to 0, whereas in cattle, we observed a higher recombination rate in males controlled by shared variants effective in both sexes. [less ▲]

The deltaretrovirus Bovine Leukemia Virus (BLV) is closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, produces a lifelong ... [more ▼]

The deltaretrovirus Bovine Leukemia Virus (BLV) is closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about ~5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. Like the case in HTLV-1 the 5’LTR BLV provirus is transcriptionally silent in tumors, however the provirus is not entirely quiescent, constitutively express the BLV microRNAs in tumors. Using RNA-seq, we found that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of ~600bp (AS1-S) and a less abundant longer transcript of ~2200bp (AS1-L). Alternative splicing also creates a second transcript of ~400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3’LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5' RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV. [less ▲]

We herein study genetic recombination in three dairy cattle populations from France, New-Zealand and the Netherlands. We identify 2,395,177 crossover (CO) events in sperm cells transmitted by 2,940 sires to 94,516 offspring, and 579,996 CO events in oocytes transmitted by 11,461 cows to 25,332 offspring. When measured in identical family structures, the average number of CO in males (23.3) was found to be larger than in females (21.4). The heritability of global recombination rate (GRR) was estimated at 0.13 in males and 0.08 in females. The genetic correlation was equal to 0.66, indicating that shared variants are influencing GRR in both genders. Haplotype-based genome-wide association studies revealed seven genome-wide significant QTL. Variants identified by next-generating sequencing in 5 Mb windows encompassing the QTL peaks were imputed in order to perform a sequence-based association analysis. For four QTLs, we identified missense mutations in genes known to be involved in meiotic recombination among the most significantly associated variants. The P259S variant identified in RNF212 had already been reported, whereas missense mutations in MLH3 (N408S), HFM1 (S1189L), MSH5 (R631Q), MSH4 (C342Y) and a second in RNF212 (A77T) are new. Surprisingly, variants previously identified in REC8 were not associated with a QTL detected on BTA10 whereas variants in RNF212B, a paralog of RNF212, showed much stronger association with the phenotype in this region. This suggests that RNF212B might be involved in the recombination process. Most of the identified mutations had significant effects in both genders with three of them accounting each for approximately 10% of the genetic variance in males (the allelic substitution effect being approximately equal to one additional CO per genome). Thus, a large fraction of the genetic variance is associated with missense mutations in genes known to be involved in meiotic recombination. Our results are very different from reports of recombination in other species. For instance, in human, recombination rate is higher in females, distinct variants affect recombination rate in males and females, and the genetic correlation is close to 0, whereas in cattle, we observed a higher recombination rate in males controlled by shared variants effective in both sexes. [less ▲]

Bovine Leukemia Virus (BLV) and Human T-cell leukemia virus-1 (HTLV-1) are closely related delta-retroviruses provoking a polyclonal expansion of infected B- and T-cells respectively, with monoclonal leukemia/lymphoma developing in about ~5% of infected individuals. To date, the molecular mechanisms leading to cellular transformation remain unclear. Both proviruses are largely transcriptionally silent in tumors and their integration sites in the host genome appear very variable. Mapping proviral insertion sites using high throughput sequencing techniques has provided insights into the evolution of BLV/HTLV-1 infections and the expansion of transformed clones in delta-retrovirus induced leukemia/lymphoma. The methods currently used have a number of limitations such as the utilisation of custom sequencing primers, relatively high sequencing costs, no examination of the 5’LTR host flanking region and a limited dynamic range for determining clone abundance. We have developed an alternative high-throughput sequencing protocol for tracking proviral integration sites and determining clonal abundance in BLV and HTLV-1 infected individuals. We implemented the use of off-the-shelf Illumina primers for the addition of adapters and indexes, which facilitates library multiplexing and avoids the need for custom sequencing primers. Our method reduces the amount of sequencing of PCR duplicates by reducing the number of PCR cycles via an enrichment of BLV- and HTLV-1-carrying DNA fragments. Moreover, in addition to the proviral 3’LTR, our approach also assays the 5’LTR, providing additional information on the frequency of 5’-end deletions in proviruses and increasing the dynamic range of the assay. We have tested the approach on over 100 BLV and HTLV-1 samples, representing both tumors and asymptomatic stages. Our approach allowed for a more accurate determination of clone abundance in tumors and by assaying the provirus 5’ end, identified clones overlooked with previously published methods. Finally, by facilitating greater multiplexing of libraries we have reduced the cost to a level where the technique may be attractive in a clinical setting. [less ▲]

We herein study genetic recombination in three cattle populations from France, New Zealand, and the Netherlands. We identify 2,395,177 crossover (CO) events in 94,516 male gametes, and 579,996 CO events ... [more ▼]

We herein study genetic recombination in three cattle populations from France, New Zealand, and the Netherlands. We identify 2,395,177 crossover (CO) events in 94,516 male gametes, and 579,996 CO events in 25,332 female gametes. The average number of COs was found to be larger in males (23.3) than in females (21.4). The heritability of global recombination rate (GRR) was estimated at 0.13 in males and 0.08 in females, with a genetic correlation of 0.66 indicating that shared variants are influencing GRR in both sexes. A genome-wide association study identified seven quantitative trait loci (QTL) for GRR. Fine-mapping following sequence-based imputation in 14,401 animals pinpointed likely causative coding (5) and noncoding (1) variants in genes known to be involved in meiotic recombination (HFM1, MSH4, RNF212, MLH3, MSH5) for 5/7 QTL, and noncoding variants (3) in RNF212B for 1/7 QTL. This suggests that this RNF212 paralog might also be involved in recombination. Most of the identified mutations had significant effects in both sexes, with three of them each accounting for approximately 10% of the genetic variance in males. [less ▲]

in American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons (2016)

Acute renal rejection is a major risk factor for chronic allograft dysfunction and long-term graft loss. We performed a genome-wide association study to detect loci associated with biopsy-proven acute T ... [more ▼]

Acute renal rejection is a major risk factor for chronic allograft dysfunction and long-term graft loss. We performed a genome-wide association study to detect loci associated with biopsy-proven acute T cell-mediated rejection occurring in the first year after renal transplantation. In a discovery cohort of 4127 European renal allograft recipients transplanted in eight European centers, we used a DNA pooling approach to compare 275 cases and 503 controls, on Illumina 2.5 M arrays. In an independent replication cohort of 2765 patients transplanted in two European countries, we identified 313 cases and 531 controls, in whom we genotyped individually the most significant SNPs from the discovery cohort. In the discovery cohort, we found 5 candidate loci tagged by a number of contiguous SNPs (>5) that was never reached in iterative in silico permutations of our experimental data. In the replication cohort, two loci remained significantly associated with acute rejection in both univariate and multivariate analysis. One locus encompasses PTPRO, coding for a receptor-type tyrosine kinase essential for B cell receptor signalling. The other locus involves ciliary gene CCDC67, in line with the emerging concept of a shared building design between the immune synapse and the primary cilium. This article is protected by copyright. All rights reserved. [less ▲]

BACKGROUND: Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a ... [more ▼]

BACKGROUND: Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about 5 % develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The mechanisms by which these viruses provoke cellular transformation remain opaque. In both viruses little or no transcription is observed from the 5'LTR in tumors, however the proviruses are not transcriptionally silent. In the case of BLV a cluster of RNA polymerase III transcribed microRNAs are highly expressed, while the HTLV-1 antisense transcript HBZ is consistently found in all tumors examined. RESULTS: Here, using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600 bp (AS1-S) and a less abundant transcript of ~2200 bp (AS1-L). Alternative splicing creates a second transcript of ~400 bp (AS2). The coding potential of AS1-S/L is ambiguous, with a small open reading frame of 264 bp, however the transcripts are primarily retained in the nucleus, hinting at a lncRNA-like role. The AS1-L transcript overlaps the BLV microRNAs and using high throughput sequencing of RNA-ligase-mediated (RLM) 5'RACE, we show that the RNA-induced silencing complex (RISC) cleaves AS1-L. Furthermore, experiments using altered BLV proviruses with the microRNAs either deleted or inverted point to additional transcriptional interference between the two viral RNA species. CONCLUSIONS: The identification of novel viral antisense transcripts shows the BLV provirus to be far from silent in tumors. Furthermore, the consistent expression of these transcripts in both leukemic and nonmalignant clones points to a vital role in the life cycle of the virus and its tumorigenic potential. Additionally, the cleavage of the AS1-L transcript by the BLV encoded microRNAs and the transcriptional interference between the two viral RNA species suggest a shared role in the regulation of BLV. [less ▲]

The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous +Mat/CLPGPat animals receiving the CLPG mutation from their father express the phenotype ... [more ▼]

The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous +Mat/CLPGPat animals receiving the CLPG mutation from their father express the phenotype. +Mat/CLPGPat animals are characterized by postnatal, ectopic expression of Delta-like 1 homologue (DLK1) and Paternally expressed gene 11/Retrotransposon-like 1 (PEG11/RTL1) proteins in skeletal muscle. We showed previously in transgenic mice that ectopic expression of DLK1 alone induces a muscular hypertrophy, hence demonstrating a role for DLK1 in determining the callipyge hypertrophy. We herein describe newly generated transgenic mice that ectopically express PEG11 in skeletal muscle, and show that they also exhibit a muscular hypertrophy phenotype. Our data suggest that both DLK1 and PEG11 act together in causing the muscular hypertrophy of callipyge sheep. [less ▲]

Four newborn purebred Belgian Blue calves presenting a severe form of epidermolysis bullosa were recently referred to our heredo-surveillance platform. SNP array genotyping followed by autozygosity mapping located the causative gene in a 8.3-Mb interval on bovine chromosome 24. Combining information from (i) whole-genome sequencing of an affected calf, (ii) transcriptomic data from a panel of tissues and (iii) a list of functionally ranked positional candidates pinpointed a private G to A nucleotide substitution in the LAMA3 gene that creates a premature stop codon (p.Arg2609*) in exon 60, truncating 22% of the corresponding protein. The LAMA3 gene encodes the alpha 3 subunit of the heterotrimeric laminin-332, a key constituent of the lamina lucida that is part of the skin basement membrane connecting epidermis and dermis layers. Homozygous loss-of-function mutations in this gene are known to cause severe junctional epidermolysis bullosa in human, mice, horse, sheep and dog. Overall, our data strongly support the causality of the identified gene and mutation. [less ▲]

We herein describe the realization of a genome-wide association study for scrotal hernia and cryptorchidism in Norwegian and Belgian commercial pig populations. We have used the transmission ... [more ▼]

We herein describe the realization of a genome-wide association study for scrotal hernia and cryptorchidism in Norwegian and Belgian commercial pig populations. We have used the transmission disequilibrium test to avoid spurious associations due to population stratification. By doing so, we obtained genome-wide significant signals for both diseases with SNPs located in the pseudo-autosomal region in the vicinity of the pseudo-autosomal boundary. By further analyzing these signals, we demonstrate that the observed transmission disequilibria are artifactual. We determine that transmission bias at pseudo-autosomal markers will occur (i) when analyzing traits with sex-limited expression and (ii) when the allelic frequencies at the marker locus differ between X and Y chromosomes. We show that the bias is due to the fact that (i) sires will preferentially transmit the allele enriched on the Y (respectively X) chromosome to affected sons (respectively daughters) and (ii) dams will appear to preferentially transmit the allele enriched on the Y (respectively X) to affected sons (respectively daughters), as offspring inheriting the other allele are more likely to be non-informative. We define the conditions to mitigate these issues, namely by (i) extracting information from maternal meiosis only and (ii) ignoring trios for which sire and dam have the same heterozygous genotype. We show that by applying these rules to scrotal hernia and cryptorchidism, the pseudo-autosomal signals disappear, confirming their spurious nature. [less ▲]

Background/Purpose: Immune-mediated inflammatory disorders (IMIDs) share many genetic risk factors. Pleiotropy may exist at different levels and most of the underlying mechanisms are still to be uncovered ... [more ▼]

Background/Purpose: Immune-mediated inflammatory disorders (IMIDs) share many genetic risk factors. Pleiotropy may exist at different levels and most of the underlying mechanisms are still to be uncovered. GWAS have identified hundreds of risk loci for IMIDs but causative genes have been identified in only a handful of cases. Recent fine-mapping efforts indicate that only a minority of risk variants are coding. This suggests that most risk variants will be regulatory hence affecting disease risk via eQTL effects. Methods: To aid in the identification of causative genes for IMIDs, we generated transcriptome information (HT12 arrays) for six blood cell types (CD4, CD8, CD19, CD14, CD15 and platelets) and intestinal biopsies at three anatomical locations (ileum, colon, rectum) for 350 healthy Caucasians. The same individuals were genotyped with SNP arrays interrogating > 700K variants, augmented by imputation from the 1KG project. To detect cis-eQTL we tested variants within 0.5 megabase windows centered on the tested probe. The nominal p-value of the best SNP within a cis-window was Sidak-corrected for the window-specific number of independent tests. The corresponding best, Sidak-corrected p-values for each probe were jointly used to estimate their respective false discovery rate.To identify likely causative genes in GWAS identified risk loci variants and also better understand pleiotropic effects, we (i) developed a method that quantifies the correlation between “disease association pattern” (DAP) and “eQTL association pattern” (EAP) and provides an empirical estimate of its significance, and (ii) evaluated the effect of fitting known risk variants as covariates in the eQTL analysis following Nica et al. (2010). We applied both approaches to celiac disease (CE) and rheumatoid arthritis (RA) and the second one to type one diabetes (T1D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), ankylosing spondylitis (AS) and psoriasis (PSO). Results: We detected > 16000 significant cis-eQTL, with a degree of sharing between cell types ranging from 38 to 90% highlighting the utility of our multi-tissue panel. GWAS variants were drivers of ciseQTL effects across the different tissues in 399 tests (23.6%), mostly in CD4 cells, and pinpointing 64 new gene-disease associations (3.7%). The number of shared loci and shared eQTL were highly correlated (rho=0.66).RA and SLE showed the highest degree of sharing. Conclusions: We identified new potential candidate genes for IMIDs and characterized pleiotropic effects through ciseQTL mapping in GWAS loci. These findings could shed a light on IMIDs pathogenesis and co-occurrence. Latest results will be presented. [less ▲]